Focal Adhesion Kinase (FAK) is an important survival molecule that is upregulated in a broad range of solid tumors and is expressed at very low levels in normal tissues, creating a therapeutic window and making this protein a highly attractive target for the treatment of cancer See, e.g., WO 2005/049852, the contents of which are incorporated by reference.
The market for novel drug therapy targeting cancers of the breast, colon, pancreas, and thyroid is extensive. According to the American Cancer Society, it is estimated that 425,000 new cases of these cancers will be diagnosed this year in this country alone. Cancer drug therapy is an existing major product line of several pharmaceutical companies, and the development of drugs targeting FAK would be a natural complement to their existing products.
FAK is overexpressed in many cancer types compared to other kinase targets. Compounds that target FAK could be prescribed for many cancer types including breast, colon, pancreas, thyroid, lung, and melanoma.
Several groups are exploring the targeting of FAK as potential cancer therapeutics. The targeting of FAK typically has been focused on the kinase domain of FAK. This approach has proven unsuccessful as disruption of the kinase domain does not specifically interfere with the signaling downstream of FAK and other related tyrosine kinases have been affected by the drugs. Delineated herein is a novel approach that focuses on FAK phosphorylation.
FAK is a 125 kDa protein that localizes to focal adhesions (1) and is activated and tyrosine phosphorylated in response to integrin clustering (2). Tyrosine 397 is an autophosphorylation site of FAK and is a critical component in downstream signaling (3), providing a high-affinity binding site for the SH2 domain of Src family kinases (4), (5). The interaction between Y397-activated FAK and Src leads to a cascade of tyrosine phosphorylation of multiple sites in FAK (-576, -577, -925), as well as other signaling molecules such as p130CAS and paxillin, resulting in cytoskeletal changes and activation of other downstream signaling pathways (6). Y397 is also a site of binding PI3 kinase, growth factor receptor binding Grb-7, Shc, and other proteins. Thus, the Y397 site is one of the main phosphorylation sites that activate FAK signaling in the cells.
Focal adhesion kinase is involved in multiple cellular functions such as cell proliferation, survival, motility, invasion, metastasis, and angiogenesis (7). Different approaches to inhibit FAK with FAK anti-sense oligonucleotides (8), dominant-negative C-terminal domain of FAK, FAK-CD or FRNK (9,10) or FAK siRNA (11), (12) caused decreased cellular viability, growth inhibition or apoptosis. Recently, FAK has been proposed to be a new potential therapeutic target in cancer (13,14). Two novel kinase inhibitors of FAK, blocking FAK catalytic activity, were developed and reported recently, one by Novartis: NVP-TAE226 (15) (16) and another by Pfizer: PF-573,228 (17). The first inhibitor, TAE226, inhibited glioma and ovarian tumor growth in vivo (16,18), although it also inhibited IGFR kinase (16). The efficacy of the PF-573,228 on tumor growth in vivo has not been reported, it inhibited only motility and did not inhibit cell growth and survival in vitro (17). Both of these inhibitors effectively blocked Y397-FAK phosphorylation.
Since the Y397 site is important for FAK survival function, we performed a computer modeling approach, described in (19), to specifically target the Y397-site of FAK and to find potential small-molecule drugs that inhibit FAK function and decrease cell viability and tumor growth.
We found that certain compounds, including 1,2,4,5-benzenetetraamine tetrahydrochloride, called Y15, targets the Y397 site, directly and specifically decreases Y397-phosphorylation of FAK in vitro, inhibits cancer cell viability in vitro, causes detachment, decreases cell adhesion and blocks tumor growth in vivo. Thus, targeting the Y397 site can be an effective therapy approach for developing future novel FAK inhibitors.